Loading Controls

Loading control proteins are known to be constitutively and stably expressed at high levels in almost all
tissues and
cells. Housekeeping proteins, such as GAPDH , beta-tubulin and beta-actin , represent some of the popular loading
control proteins. Loading control antibodies help determine whether samples have been loaded equally in each
lane.
This information is needed to determine whether the difference in levels of the protein of interest in
different
samples is due to loading variance or sample-to-sample expression level differences. View more about loading control FAQs.

Why Choose OriGene Loading Controls

b. Most are monoclonal antibodies provided under rigorous quality control on a panel of cell lines.

c. HRP- and biotin-conjugated versions of these antibodies are also available.

Loading Control Background

Beta-actin is an isform of actin proteins. Actins are highly conserved
proteins that are involved in cell motility, structure, and integrity. There are six different but highly conserved
actin isoforms in vertebrates. Four of these isoforms are expressed primarily in striated and smooth muscle cells,
whereas the two cytoplasmic β-actin and γ-actin isoforms are ubiquitously expressed. Conserved from birds to
mammals, β-actin and γ-actin differ at only four biochemically similar amino acid residues. These amino acid
differences confer different biochemical properties between the two isoforms. Beta-actin is a major constituent of the
contractile apparatus.

Beta-actin, also known as a "housekeeping" protein, is usually used as a loading control, for among others, the
integrity of cells, protein degradation, in PCR and Western blotting. Loading controls are essential for proper
interpretation of western blots. They can be used to normalize the levels of protein detected by confirming that
protein loading is the same across the gel.

Beta-tubulin , is a subunit of tubulin. Tubulin is one of several
members of a small family of globular proteins. It is the major constituent of microtubules. There are two of the most common members of the tubulin family: alpha-tubulin and beta-tubulin, and together their dimers form microtubules.
The dimers of alpha- and beta-tubulin bind to GTP and assemble onto the (+) ends of microtubules while in the
GTP-bound state. After the dimer is incorporated into the microtubule, the molecule of GTP bound to the beta
-tubulin subunit eventually hydrolyzes into GDP through inter-dimer contacts along the microtubule protofilament.
Beta-tubulin faces the plus end of the microtubule while alpha-tubulin faces the minus end. Dimers bound to GTP tend
to assemble into microtubules, while dimers bound to GDP tend to fall apart.

GAPDH, less commonly as G3PDH, is the abbreviation of Glyceraldehyde
3-phosphate dehydrogenase. It is an enzyme of ~37kDa that catalyzes the sixth step of glycolysis and thus serves to
break down glucose for energy and carbon molecules. GAPDH is considered a housekeeping gene because this gene is
often stably and constitutively expressed at high levels in most tissues and cells. So GAPDH is commonly used by
biological researchers as a loading control for western blot and as a control for qPCR.

Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample you
are examining. This ensures constant expression levels. Thus "housekeeping genes" are frequently chosen for use as
loading controls. It is also important that the protein chosen as a loading control has a different molecular weight
with the protein of interest so that the bands are distinct and expression levels quantifiable. Popular loading
control detection antibodies include anti-β-actin monoclonal or polyclonal antibodies, anti-COX-4, anti-GAPDH,
anti-Tubulin and anti-VDAC/Porin antibodies.